Novel and practical serological assay for the clinical diagnosis of Leishmaniasis

ABSTRACT

Methods for the diagnosis of visceral, cutaneous and canine leishmaniasis in a subject suspected of being infected with the parasitic protozoa Leishmania is disclosed. Disclosed are antibody-capture enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies to Leishmania parasite soluble antigens and antigen-capture ELISAs for the detection of Leishmania parasite soluble antigens in host samples. Also disclosed are immunodiagnostic kits for the detection of Leishmania parasite circulating antigens or IgM and IgG antibodies in a sample from subject having visceral, cutaneous or canine leishmaniasis. In these methods and kits, detection may be done photometrically or visually. The methods and kits also allow the visualization of Leishmania amastigotes or promastigotes in a sample.

RELATED APPLICATION DATA

This application claims the benefit of U.S. Provisional PatentApplication No. 60/168,300, filed Dec. 1, 1999, naming Jeffrey R. Ryan,Samuel K. Martin, and Anthony M. Smithyman as co-inventors, which isherein incorporated by reference.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

This invention was made by employees of the United States Army. Thegovernment has rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to a method of diagnosing leishmaniasisin a subject suspected of being infected with the parasitic protozoaLeishmania. In particular, the invention relates to enzyme-linkedimmunosorbent assays (ELISAs) for the detection of Leishmania parasitecirculating antigens and Leishmania-specific antibodies in host samples.

2. Description of the Related Art

Leishmaniasis is a serious and sometimes fatal disease estimated by theWorld Health Organization (WHO) to affect approximately 12 millionpeople in 88 countries. Recent epidemics of leishmaniasis have occurredin the Africa, the Indian subcontinent and Brazil. In addition to theconcerns regarding significant morbidity and mortality caused byleishmaniasis in endemic areas, the increase in global travelconcomitant with the increased incidence of the disease in HIV-infectedand intravenous drug-user populations is a cause for concern innon-endemic areas more recently.

Unfortunately, current acceptable diagnostic practices lack the meansfor efficiently and accurately identifying those infected or exposed tothe disease-causing parasite as explained in Martin, S. et al. (1998)Military Medicine 163(23):801-807. As a result, the prevention ofleishmanial epidemics is greatly hindered and patient management isdifficult. Additionally, there is an imminent threat of a developingpopulation of people co-infected with HIV and Leishmania. To date, thereare no antigen-detection type diagnostic tests available forLeishmaniasis. In view of this, antigen-detection assays are desperatelyneeded for diagnosis, patient management and epidemiological studies.

Recombinant kinesin protein, rK39, is one of the few antigens that hasbeen used in the development of antibody-detection immunoassays foractive visceral leishmaniasis (VL). However, assays of this antigen andothers fail to consistently detect antibodies in other clinicalsyndromes associated with a predominately T-cell and muted B-cellresponse. Moreover, antibody-detection assays have an inherentlimitation—dependence on the immune response of the host to the parasiteantigen. For example, in an immunocompromised host, the infection maynot produce proportional antibody production and thereby escapedetection.

Generally, the serological tests for diagnosing VL are highly sensitive(>90%). See Senaldi, G., et al., (1996) J. Immunol. Methods 193:9-5.These serological tests, however, pose problems of non-specificityresulting in false positive results from reference samples of otherinfectious diseases and subclinical leishmanial infections.Modifications of the antigens used for direct agglutination test (DAT)and for ELISA are reported to be markedly successful in eliminatingfalse-positive results. See Zijlstra, E. E., et al., (1997) Trans. R.Soc. Trop. Med. Hyg. 91:671-673 and Badaro, R., et al., (1996) J. Inf.Dis. 173:758-761. Unfortunately, serological tests are rarely performedto diagnose cutaneous leishmaniasis because the sensitivities andspecificities are disappointingly poor for that clinical manifestation.See Sanchez, J. L., et al., (1992) Am. J. Trop. Med. Hyg. 47:47-54;Garcia-Miss, M. R., et al., (1990) Trans. R. Soc. Trop. Med. Hg.84:356-358.

The antigens used in immunoassays for the detection of leishmaniasis aretraditionally derived from promastigotes cultivated in vitro, or fromrecombinant proteins. See Badaro et al. (1996); Choudhary, S., et al.,(1992) J. Comm. Dis. 24:32-36; Badaro, R., et al., (1986) Am. J. Trop.Med. Hyg. 35:72-78; Choudhary, A., et al., (1990) Trans. R. Soc. Trop.Med. Hyg. 84:363-366; and Reed, S. G., et al., (1990) Am. J. Trop. Med.Hyg. 43:632-639. The promastigotes release metabolic products into theculture medium to produce conditioned medium. These metabolic productsare immunogenic to the host. See Schnur, L. F., et al., (1972) Isrl. J.Med. Sci. 8:932-942; Sergeiev, V. P., et al., (1969) Med. Parasitol.38:208-212; El-On, J., et al., (1979) Exper. Parasitol. 47:254-269; andBray, R. S., et al., (1966) Trans. R. Soc. Trop. Med. -Hyg. 60:605-609.

The prior art assays are based on these metabolic products found in thein vitro culture medium. Thus, the presence of complex proteins or serumcomponents required for growth of the parasites in culture pose severalproblems in the prior art assays. For example, the amount ofmanipulation needed to purify the targeted antigens from the metabolicproducts can affect the native composition of certain componentsnecessary for a highly sensitive assay. Furthermore, insufficientpurification techniques may create problems with specificity as commonproteins remain in the antigen preparations, which cause non-specificreactions. Generally, the prior art assays are limited in scope to onespecies complex or clinical manifestation and have never demonstratedcombined sensitivity and specificity of more than 90%.

Thus, a need exists for highly sensitive and highly specific assays fordetecting, measuring, or diagnosing exposure to Leishmania.

SUMMARY OF THE INVENTION

The invention relates to an immunoassay for detecting IgM and IgGantibodies in a sample from a subject having visceral, cutaneous orcanine leishmaniasis.

The invention also relates to an immunoassay for detecting Leishmaniaparasite circulating antigens in a sample from a subject havingvisceral, cutaneous or canine leishmaniasis.

In one embodiment, the invention relates to a leishmaniasis immunoassay,which is based on soluble antigens from promastigotes cultivated in aprotein-free and serum-free medium.

In another embodiment, the invention relates to the use of protein-freeand serum-free medium to cultivate the promastigotes in an immunoassayfor the diagnosis of leishmaniasis.

In a preferred embodiment, the assay of the invention has a high degreeof sensitivity and specificity. For example, the assay of the inventionhas a combined sensitivity and specificity of more than about 90%.Preferably, the assay of the present invention has sensitivity of about95.1% and a specificity of about 100%.

The invention also relates to an antigen-capture immunoassay for thedetection of exo-antigens released by the parasite in a sample fromsubject having visceral, cutaneous or canine leishmaniasis.

The invention also relates to immunodiagnostic kits for the detection ofspecific IgM and IgG antibodies to Leishmania parasite circulatingantigens in a sample from a subject having visceral, cutaneous or canineleishmaniasis. Detection may be done photometrically or visually. Ifvisual detection is desired., a fluorescent. chromogenic orchemiluminescent agent may be utilized. Preferably, the intensity oramount of the visual signal is in proportion to the amount of theantibody present in the sample.

In one embodiment, the invention allows the visualization of Leishmaniaamastigotes or promastigotes by the use of a fluorescein conjugatedpolyclonal antibody to the specific antigen preparation aforementioned.

Preferably, the protein-free and serum-free medium utilized in thepresent invention comprises a cross-linking agent that is notmetabolized by cultures and is relatively easy to purify out bydialysis. In a preferred embodiment of the invention, the mediumcomprises xylose as xylose is not metabolized by cultures. Additionally,only small amounts of xylose are required. Since only small amounts ofxylose are necessary, purification is relatively easy and lessdestructive to the components found in the conditioned medium.

DESCRIPTION OF THE DRAWINGS

This invention is further understood by reference to the drawingswherein:

FIG. 1 is a boxplot to illustrate specific IgG antibody levels measuredin VL patient sera samples.

FIG. 2 is a boxplot showing specific IgM antibody levels measured in thesame samples.

FIG. 3 is a boxplot illustrating specific IgG antibody levels measuredin CL patient sera samples.

FIG. 4 is a boxplot depicting specific IgM antibody levels measured inthe same samples.

FIG. 5 shows specific IgG antibody levels measured in canineleishmaniasis sera samples.

FIG. 6 is a boxplot that displays specific IgM antibody levels measuredin the same samples.

FIG. 7 is a Western Blot illustrating the striking rabbit pattern (lanes1 and 2) similar to that of the kala azar patients pattern (lanes 5 and6).

FIG. 8 is a graph illustrating the specific activity of the competitiveAg-capture ELISA while using different antigen and HRP-labeled antigenconditions.

FIG. 9 is a graph illustrating the specific activity of the competitiveAg-capture ELISA while titrating labeled antigen under different coatingand blocking conditions.

DETAILED DESCRIPTION OF THE INVENTION

Recently, the soluble antigens shed, excreted and secreted by Leishmaniaparasites in a protein-free medium have been described. The solubleantigens of L. donovani promastigotes, are primarily lipophosphoglycan(LPG), and comprise an albumin binding site, a hydrophylic LPGcomponent, and a repeating phosphorylated saccharide linked withsecreted acid phosphatase (S-AcP). See Greis, K. D., et al., (1992) J.Biol. Chem. 267:5876-5881. S-AcP is presumably the most immunogenic ofall the glycoproteins and is a major component in L. donovanipromastigote conditioned medium. See Bates, P. A., et al., (1988) Exper.Parasitol. 67:199-209. Monoclonal antibody (MAb) studies show no crossreactivity between S-AcP and parasite surface membrane or host acidphosphatases. See Bates, P. A., et al., (1987) Exper. Parasitol.64:157-164. S-AcP from L. donovani promastigote conditioned medium hasbeen used to immunoprecipitate specific antibody from pooled sera ofpatients acutely ill with VL (kala-azar). See Ellis, S. L., et al.,(1998) Exp. Parasitol. 89:161-168.

The use of a soluble antigen preparation from Leishmania donovani tocapture specific IgG antibodies in the sera of kala-azar patientsindicates that the soluble antigens found in conditioned medium can actas the foundation for leishmaniasis immunoassays. See Martin et al.(1998) An. Trop. Med. & Parasit. 92: 571-577.

Thus, the present invention generally relates to leishmaniasisimmunoassays, which are highly sensitive and specific and allow thedetection of specific IgG and IgM antibodies in subjects affected withvisceral, cutaneous, or canine leishmaniasis. The present invention alsorelates to antigen-capture immunoassays which allow the detection of thesoluble Leishmania antigens.

The present invention relates to an immunoassay that allows thedetection of IgM and IgG antibodies in subjects affected with visceral,cutaneous, or canine leishmaniasis. The assay is based on solubleantigens from promastigotes cultivated in a protein-free and serum-freemedium and takes less than four hours to perform.

Prior research attempts employing non-recombinant antigens in the designof serologic tests for leishmaniasis have been limited by problems withsensitivity, specificity and test reproducibility. Reasons for theselimitations remain elusive, but are most likely attributable to physicaland chemical techniques used in antigen preparation. Therefore, thepresent invention relates to sensitizing plates with soluble antigensfrom conditioned media. Plate sensitization ameliorates the prior artproblems associated with physical and chemical techniques used inantigen preparation.

In order to obtain soluble antigens from conditioned media, cells mustbe maintained within defined physiological ranges to thrive in vitro.These conditions include temperature, pH, osmotic pressure, O₂ and CO₂gas tension, and nutrients. It has been shown that cells fail to thrivein culture media unless 10 to 20% serum albumin is added and theviability and growth rates are compromised where serum albuminconcentrations vary substantially from 10%. It is believed that theprimary role of serum albumin in in vivo and in vitro cell culturesystems is to balance oncotic pressure across the semi-permeablemembrane of cells and to provide free water homeostasis. It is alsobelieved that the critical component of serum for in vitro cell survivalis albumin.

Therefore, one aspect of the present invention is the propagation of thepromastigotes in serum-free and protein-free medium comprising an agentthat balances the oncotic pressure across the semi-permeable membrane ofthe cells. An example of this agent is xylose. Thus, the uncharacterizedsoluble factors in conditioned medium from the cultivation ofpromastigotes in protein-free and serum-free medium can be used as afoundation for an immunoassay for visceral, cutaneous, or canineleishmaniasis. The use of a defined protein-free and serum-free culturemedium reduces antigen production to a simple and inexpensivecentrifugation step and greatly increases the sensitivity, specificity,reproducibility and practicability of the assay. Additionally, gramquantities of the soluble antigens may be inexpensively and reproduciblygenerated by methods standard in the art. Furthermore, reactivity to thesoluble antigens appears to be genus specific.

Since sensitivity to oncotic imbalance varies with cell type, one maywish to conduct experiments to determine the optimum concentration rangeof the agent which balances the oncotic pressure across thesemi-permeable membrane of a given cell type. For example, parasites maybe cultured in a serum-free and protein-free medium, wherein a colloidalagent balances the oncotic pressure. The growth index may be determinedby comparing the growth of the parasites in the serum-free andprotein-free medium with the growth of the parasites in serumsupplemented media. Based on the comparison, one may then choose theoptimum concentration range of the colloidal agent.

When attempting to detect specific IgM, normally one must consider theeffect of IgG in the sample and compensate for its presence or eliminateit altogether. Thus, a fraction of samples tested (n=100) were subjectedto a Rapi-Sep spin column (INDX), which binds the IgG in the samples tothe membranes, before testing for specific IgM. There was no significantchange in the IgM values derived for any sample.

In preliminary studies, 129 visceral (Brazil, Italy, North Africa,Nepal) and 143 cutaneous (Brazil) leishmaniasis patients with controlswere tested. These studies show an overall sensitivity of 95.1% whenLeishmania-specific IgG was measured against that of healthy, NorthAmerican negative controls. No cross-reactivity was noted when thisassay was used to look for cross-reacting antibodies in patient samplesfrom other parasitic diseases (malaria, echinococcosis, Africatrypanosomiasis, and filariasis). See Martin et al.

In additional studies, the assay of the present invention correctlyidentified 42 sera from Brazilian dogs with canine leishmaniasis and 10healthy controls. As shown in FIGS. 1-6, the difference between negativeand positive was greater in the case of dog sera than of human sera. Thenegative control sera subset gave a negative cutoff score of about 0.225for the IgG assay. FIG. 1 is a boxplot to illustrate specific IgGantibody levels measured in VL patient sera samples. With respect tospecific IgG, all 129 clinically confirmed positive VL patient sera gaveOD readings above the negative cutoff (100% sensitivity). FIG. 2 is aboxplot showing specific IgM antibody levels measured in the samesamples. When measuring IgM, the negative control sera tested gave anegative cutoff score of about 0.310. The sensitivity for this assay was94.57% (122/129 positive).

Currently, the assay is able to detect specific IgG and IgM antibodieswith varying degrees of success in patient serum samples fromknown-positive cases of visceral leishmaniasis (VL) and cutaneousleishmaniasis (CL). The assay may be improved by modifying serum sampleand conjugate dilution, substrate, incubation times, temperatures, andother assay conditions, such as described in Example 8, and is wellwithin the skill and knowledge of one of ordinary skill in the art. Forexample, the serum may be diluted by 1:1000 instead of 1:200 and allincubations may be at room temperature instead of a 37° C. humiditychamber. Serum samples may be diluted from about 1:250 to about 1:1,000.Preferably, for CL assays, the serum sample is diluted to 1:250 and forVL assays, the serum sample is diluted to 1:1,000. The wide range ofconjugates that may be produced from polyclonal antibodies against theseantigens vary in their affinity and avidity. As such, their workingdilutions range from 1:5,000 to about 1:32,000. However, one of ordinaryskill in the art may readily determine the optimum dilutions for a givensample and assay by methods standard in the art such as checkerboardtitrations.

Initially, all categories of patient samples were tested with theWR0130E L. donovani antigen (ATCC strain 30503). However, the assay wasnot sensitive to CL patient sera samples. Thus, these CL samples wereretested with the L. mexicana (ATCC strain 50157) antigen.

The immunoassay of the present invention may detect IgM and IgGantibodies in human patients with visceral and cutaneous leishmaniasis,and dogs with canine leishmaniasis. When using Leishmania-specific IgGantibodies as a marker for active disease the test showed an overallsensitivity of 95.9% (261/272) and a specificity of 100%.

The following examples are intended to illustrate but not to limit theinvention.

EXAMPLE 1 Sera Preparation

Serum was collected from human patients who were admitted to clinics inBrazil, Italy, North Africa, Nepal and Walter Reed Army Medical Centerand who had either splenic aspirates or skin biopsies from lesions thattested positive for Leishmania parasites by culture and microscopy. Intotal, 129 visceral (Italy, Brazil, North Africa, and Nepal) and 143cutaneous (Brazil) leishmaniasis patients (136-L. braziliensis-infected,and 7 L. panamensis-infected) with controls were tested.

Human negative controls were from 12 non-endemic area normal patientswith no documented infection or exposure to Leishmania parasites. Inaddition to the human manifestations assayed, sera from 42 Braziliandogs with a clinical diagnosis for canine leishmaniasis were testedagainst positive control sera from a commercial source (Bordier AffinityProducts, S. A., Crissier, Switzerland) and 10 negative controls from apathogen-free, canine research colony (College of Veterinary Medicine,North Carolina State University, Raleigh, N.C., USA).

EXAMPLE 2 Antigen Preparation

The Leishmania soluble antigen (exo-antigen) preparation was made bycultivating Leishmania promastigotes in normal supplemented media (RPMI,MEM plus FBS) at 26° C. until the culture reached mid-log phase at adensity of about 10⁹ cells/ml. Then the cells were pelleted and washed 6times in a defined, protein-free medium such as XOM available from GIBCOBRL, formula number 96-0051DJ, RPMI Medium 1640 comprising D, xylose at0.076 mM, Hepes buffer at 25 mM, L-glutamine, and sodium bicarbonate at30 mM without phenol red.

The cells were then resuspended in protein-free medium such as XOM to afinal density of 10⁸ promastigotes/ml and incubated at 26° C. in aroller bottle with 0.01% Tween 80 (Sigma Chemical Co., St. Louis, Mo.)for 72 hours. The cells were pelleted by centrifugation at 9,000×g for30 minutes and the supernatant was collected. The relative proteinconcentration of the soluble antigens was estimated by measuring theoptical density at 280 nm. The antigen may be stored at 4° C.

EXAMPLE 3 Antibody and Conjugate Production

The leishmania soluble antigen preparation produced by the methodexplained in Example 2 was used without an adjuvant to immunize rabbits.The antiserum was pooled and affinity-purified on a Protein A columncontaining the antigen preparation of Example 2 above. Fractions of thepolyclonal antibody (PAb) were conjugated with an appropriate reportersystem such as horseradish peroxidase, fluorescein and colloidal gold.These tagged antibodies may then be used in antigen-detectionimmunoassays such as ELISA, histochemical stain and dipstick testformats. These test formats may be used to detect parasite antigens intissues and body fluids of mammalian hosts and vectors.

The rabbit anti-leishmania polyclonal antibody preparation demonstratedhigh affinity and avidity for the immunogens used to immunize therabbits. The PAb produced a very striking Western Blot pattern similarto that of kala azar patients. See FIG. 7.

It is appreciated that the present invention also encompasses monoclonalantibodies against leishmania soluble antigens, hybridomas producingsuch, and methods of making and using thereof. Thus, monoclonalantibodies against leishmania soluble antigens may be used in the assaysdescribed herein. The monoclonal antibodies may be made by standardmethods known in the art.

EXAMPLE 4 Antibody-Capture Enzyme-Linked Immunosorbent Assay

Generally, in the solid phase enzyme immunoassay for Leishmania, solubleantigens of the Leishmania promastigotes were coated on the innersurface of a test well which serve to bind specific antibody from asample. Peroxidase conjugated antibody to anti-human IgG was added andreacted with bound antibody. A chromogenic substrate, such ashorseradish peroxidase, for peroxidase was added. If antibody toLeishmania was present, there was a reaction that resulted in thedevelopment of color. Other fluorescent, chemiluminescent andchromogenic agents may be used with appropriate enzymes and substrates.

Plate sensitization was affected by coating a polystyrene, 96-wellmicrotitre plates (Immulon 4, Dynatech Laboratories, Chantilly, Va.)with 100 μl of the respective exo-antigen solution (5 μg protein perwell). L. donovani (Walter Reed reference strain 130, clone E)exo-antigen was used to sensitize plates for visceral and canineleishmaniasis samples, and L. mexicana (ATCC strain 50157) exo-antigenwas used to sensitize plates for cutaneous leishmaniasis samples.Positive and negative controls were diluted at the same ratio as thesample. The dilutions were then placed in the wells of the microtitreplate. The samples were covered and incubated for 1 hour at roomtemperature in a humid environment.

Each plate was then blocked with 1.0% casein (Sigma Chemical Co., St.Louis, Mo.) in PBS for one hour at room temperature. The blocking bufferwas removed by aspiration and the serum samples (100 μl of 1:1000dilution) and appropriate controls were added to the microtiter plateand the plate was incubated at 26° C. for 40 minutes. The plate waswashed with 0.05% PBS-Tween-20 (PBS-Tween) buffer four times.Preferably, an automatic plate/strip washer is used. The well contentswere shaken out at the end of the final wash. Then goat anti-human IgG(whole molecule) conjugated with horseradish peroxidase (Kirkegaard &Perry Laboratories Inc., Gaithersburg, Md.) was added at 1:5000 dilutionand then the plate was incubated at 26° C. for an hour in a humidenvironment.

The plate was then washed four times with PBS-Tween buffer and 100 μl ofTMB substrate (KPL. Inc., Gaithersburg, Md.) was added to each well. Theplate was incubated for about 15 minutes in the dark. The opticaldensity (OD) was periodically read at 650 nm wavelength in an ELISAplate reader (Molecular Devices, Menlo Park, Calif.) until the OD valueof a reference positive control (S5, Kala azar patient, Nepal) reached0.8. At this point 100 μl of a stop solution (0.1M phosphoric acid) wasapplied to each well and the final OD reading was taken immediately at450 nm. Preferably, a dual beam ELISA reader is used.

A reference positive serum was used in all plates, and only interassayvariation of less than 10% was accepted. The lower limit of positivity(cut off) was determined by the mean of the negative controls subset+3standard deviations.

A. Visceral Leishmaniasis.

Generally, for the test results to be accepted for visceral leishmania,the negative control must have an OD reading under 0.15 and the positivecontrol must be over 0.8 at 650 nm. If the controls do not satisfy thiscriteria, the test should be repeated. Samples yielding absorbancevalues under 0.2 are negative and samples above 0.2 but below 0.3 maycontain antibody but the amount is lower than the generally acceptedsignificant level. It is noted that one may designate a more stringentor less stringent range for determining the absorbance levels that areindicative of exposure to leishmaniasis antigen. Samples givingabsorbency values above 0.3 contain higher levels of antibody that aregenerally considered to be at or above the significant level.

L. donovani WR0130E (ATCC strain 30503) exo-antigen was used as thematerial to coat the microtitre plate. The negative control sera subsetgave a negative cutoff score of about 0.225 for the IgG assay. Withrespect to specific IgG, all 129 clinically confirmed positive VLpatient sera gave OD readings above the negative cutoff (100%sensitivity). When measuring IgM, the negative control sera tested gavea negative cutoff score of about 0.310. The sensitivity for this assaywas 94.57% (122/129 positive).

B. Cutaneous Leishmaniasis.

Generally, for the test results to be accepted for cutaneousleishmaniasis, the negative control must have an OD reading under 0.3and the positive control must be over 0.8 at 650 nm. If the controls donot satisfy these criteria, the test should be repeated. Samplesyielding absorbance values under 0.3 are negative and samples above 0.3but below 0.3 may contain antibody but the amount is lower than thegenerally accepted significant level. Again it is noted that one maydesignate a more stringent or less stringent range for determining theabsorbance levels that are indicative of exposure to leishmaniasisantigen. Samples giving absorbance values above 0.3 contain higherlevels of antibody that are generally considered to be at or above thesignificant level.

L. mexicana exo-antigen (ATCC strain 50157) was used as the material tocoat the microtitre plate. The negative control sera subset gave anegative cutoff score of approximately 0.3 for the IgG assay. Theboxplot in FIG. 3 illustrates specific IgG antibody levels measured inCL patient sera samples. With respect to specific IgG, 132/143clinically confirmed positive CL patient sera gave OD readings above thenegative cutoff (92.31% sensitivity). FIG. 4 is a boxplot depictingspecific IgM antibody levels measured in the same samples. Whenmeasuring IgM, the negative control sera tested gave a negative cutoffscore of approximately 0.15. Only a few samples (n=6) were dramaticallyabove the negative cutoff score, the majority of values for positivesamples at or near the median value of the negative control subset. Thisassay failed to consistently detect specific IgM in CL patient serasamples (37.9%; 22/58 positive).

C. Canine Leishmaniasis.

L. donovani WR0130E (ATCC strain 30503) exo-antigen was the materialused to coat the microtitre plate. The negative control sera subset gavea negative cutoff score of an OD reading of about 0.1 at 650 nm for theIgG assay. FIG. 5 shows specific IgG antibody levels measured in canineleishmaniasis sera samples. With respect to specific IgG, all 41clinically confirmed positive canine leishmaniasis sera samples gave ODreadings above the negative cutoff (100% sensitivity). Note therelatively large degree of separation between the positive and negativecontrol subsets. The boxplot in FIG. 6 displays specific IgM antibodylevels measured in the same samples. When measuring IgM, the negativecontrol sera tested gave a negative cutoff score of approximately 0.25.The sensitivity for this assay was 97.56% (40/41).

SDS-PAGE and Western Blot analysis confirmed the ELISA results for bothVL and CL patients, canine and all negative controls. Mini-Protean II(Bio-Rad, Hercules, Calif.) was used for SDS-PAGE. Each antigenpreparation was boiled for 5 minutes in sample buffer without a reducingagent and was immediately subjected to electrophoresis on 4% stacking,12.5% separating bis-acrylamide gels. A wide-range molecular mass marker(Bio-Rad, Hercules, Calif.) was used. An antigen load of 120 μg ofprotein was used in each mini-gel. The gels were run at 100 V ofconstant voltage for 1.5 hours in Tris-glycine-SDS buffer (pH 8.3).

Protein bands from the gel were transferred to nitrocellulose. Antigensfrom the SDS-polyacrylamide gels were electroblotted onto 0.45 μm poresize nitrocellulose membranes (Bio-Rad, Hercules, Calif.) with standardtransfer buffer (0.02 M Tris, 0.15 M glycine, 0.1% SDS, 20% methanol)and 250 mA of constant current for 1 hour at 4° C. Following theblotting, portions of the membranes containing the protein markers werestained with 0.5% Amido black for 5 minutes and were destained indistilled water—glacial acetic acid solution. The membranes wereimmediately blocked with a 2% skim milk TTBS (100 mM Tris, 0.9% NaCl,0.1% Tween 20) solution and kept refrigerated until use.

Detection of antibodies from subject sera bound to the antigens of theWestern blot was done with an avidin-biotin-alkaline phosphatase systemby methods standard in the art. Strips 4 mm wide were cut frompreviously blotted and blocked membranes. These strips were incubatedwith diluted sera (1:3,200 in TTBS) for 30 minutes at room temperaturewith constant agitation. After incubation with the primary antibody, thestrips were washed 4 times for 10 minutes each time with TRBS. After thelast wash, biotinylated, anti-human immunoglobulin G was added and themixture was incubated for 30 minutes under the same conditions asdescribed above. Preformed avidin-biotin-alkaline phosphatase complexwas added. The mixture was then incubated under the same conditions asabove. The membranes were developed with a BCIP/NBT substrate(Kirkegaard & Perry Laboratories, Gaithersburg, Md.) for 5 minutes. Thereaction was stopped by rinsing the strips with distilled water and thenadding PBS-EDTA (20 mM).

EXAMPLE 5 Antigen-Capture Enzyme-Linked Immunosorbent Assay

A. General Antigen-Capture ELISA.

An antigen-capture ELISA (“sandwich” ELISA) format based on the samesoluble antigens and their complementary antibodies was developed todetect active infection in vertebrate hosts and sand fly vectors.

The capture polyclonal antibody was adsorbed to the wells of amicrotiter plate. After the capture polyclonal antibodies were bound tothe plate, the well contents were aspirated and the remaining activebinding sites on the plates were blocked with blocking buffer. A samplesuch as patient sera, urine, or ground sand flies was then tested. Testsamples were appropriately diluted with blocking buffer and an aliquotwas tested. Sand flies to be tested were ground in blocking buffer withNonidet P-40 (ELISA grade Sigma casein, bovine milk) and an aliquot wastested. Positive and negative controls were also added.

If parasite antigen was present it formed antigen-antibody complexeswith the polyclonal sera used to coat the plate. After a 2-hourincubation, the sample was aspirated and the wells were washed. Theperoxidase-linked polyclonal sera were then added to the wells, therebycompleting the formation of the sandwich. Other chromogenic agents suchas colloidal gold and FITC may be used with their correspondingsubstrates.

After 1 hour, the well contents were aspirated, the plate was washed anda clear peroxidase substrate solution from Kirkegaard & PerryLaboratories (Gaithersburg, Md.) was added. As the peroxidase enzymereacted with the substrate a dark product was formed, which theintensity of its color was relative to the amount of circulating antigenpresent in the test sample. Quantitative results were obtained by makingan endpoint determination a few minutes after the substrate has beenadded by measuring the optical density of the well contents at 450 nmwith an ELSIA plate reader. However, qualitative results may be readvisually in the field. ELISA positive samples may be retested to confirmpositives and to estimate the amount of circulating antigen per sample.

Recently, the antigen-specific, conjugated polyclonal antibodies wereused in combination with unconjugated PAb and monoclonal antibodies(MAb) specific for leishmanial Secretory Acid Phosphatases (S-cAcP) todevelop a simple antigen-capt capture assay. The sensitivity of thisassay was tested with serial dilutions of the antigen preparationprepared from the method explained above. Specific activity was recordedwith the use of an HRP conjugate at a 1:32,000 dilution. The level ofsensitivity in measuring these antigens with PAb was 400 ng/ml.

B. Rapid Wicking Assay.

A rapid wicking assay, based on the dual “sandwich” ELISA, wasdeveloped. The assay is conducted by placing a dipstick impregnated withimmobilized polyclonal antibodies from rabbits immunized with thesoluble antigen (Cellabs Pty, Ltd. Sydney, Australia) into a testsolution. When soluble leishmanial antigen is present in the solution,it binds to a specific antibody with a gold sol particle label. As theantigen-antibody-gold complexes migrate through a test zone on thedipstick comprising immobilized polyclonal antibodies from rabbitsimmunized with the soluble antigen (Cellabs Pty, Ltd. Sydney,Australia), they bind to the corresponding immobilized antibodies toform a “sandwich”. The unbound dye complexes migrate out of the testzone and can be captured later in a control zone. A reddish-purple linedevelops in the specific area of the test zone when antigen is present.A control line in the control zone should develop provided that the testwas conducted correctly.

The test zone comprises immobilized polyclonal antibodies from rabbitsimmunized with the soluble antigen (Cellabs Pty, Ltd. Sydney,Australia). The control zone comprises antibodies to immobilizedpolyclonal antibodies from rabbits immunized with the soluble antigen(Cellabs Pty, Ltd. Sydney, Australia). Monoclonal antibodies against thesoluble antigen may be used.

The test samples may be sand flies or other organisms comprisingleishmania parasites ground up in a solution such as PBS with 0.5%casein. Alternatively the test samples may be blood, serum, urine,mucus, tears, stool or the like, obtained from a subject. When the testsample is urine, it is preferably undiluted.

EXAMPLE 6 In vitro Direct Immunofluorescence Test

Fluorescein-labeled polyclonal antibodies raised against specificantigens of Leishmania parasites may be used in an in vitro directimmunofluorescence assay. The labeled antibody binds specifically to theantigens present on the surface of the parasite which can be detected ina variety of smears such as vector specimen, in vitro culture materialand patient biopsy smears.

Smears were prepared on glass slides and fixed with methanol. Unboundantibodies were removed by washing. When viewed under a fluorescencemicroscope, Leishmania parasites were seen as bright apple-greenorganisms characteristic to their life cycle stages contrasted with thereddish brown color of counterstained material. Promastigotes in thevectors or in culture were detected by their characteristic long andslender body (about 20 μm in length) with an anterior flagellum.Amastigotes present in clinical samples were detected by theircharacteristic round or oval shape measuring about 2-5 μm in diameter.

Generally, sample smears were prepared on slides marked with wells or onplain glass slides. Suitable smears had adequate specimen and weremoderately thin. For sample smears prepared on a single well slide, anadequate amount of fluorescein-labeled purified polyclonal antibodydiluted in a protein stabilized buffer solution (pH 7.4) with Evans Blueas a counter stain and 0.1% w/v sodium azide was added to the fixedsample smear and positive control.

For smears prepared on plain glass slides, after drying, the smear waspretreated by dipping the slide in a Coplin jar containing 0.1% sodiumdeoxycholate prepared in 0.85% NaCL for 5-10 minutes. The smear wasair-dried. Then an adequate amount of fluorescein-labeled purifiedpolyclonal antibody diluted in a protein stabilized buffer solution (pH7.4) with Evans Blue as a counter stain and 0.1% w/v sodium azide wasadded to the fixed sample smear and positive control.

The slides were incubated at 37° C. in a moist chamber for 30 minutes inthe dark. Then the slides were rinsed in a saline bath for about 2-5minutes. The slides were allowed to air dry and then mounted withcoverslips. With a fluorescence microscope under oil immersion, theslides were read.

This test format was found to be very sensitive to detecting amastigotesin infected patient tissues from subjects with cutaneous leishmaniasisinfected with L. brasiliensis and splenic aspirates from subject withvisceral leishmaniasis infected with L. donovani. It may be used tohighlight surface antigens and cellular structure in culturedpromastigotes. Preliminary results from gene cloning experiments withthe L. donovani clone used to produce the exo-antigen indicate that oneof the major immunogens in the sensitization of the rabbits used togenerate the PAb is a major surface antigen.

EXAMPLE 7 Competitive ELISA

Microtiter plates were coated with the anti-leishmania PAb and blockedwith 1% yeast extract. The exo-antigen was labeled with HRP. Theresulting exo-antigen conjugate was mixed 1:1 with samples and appliedto the plate and incubated overnight at 4° C. The plate was washed 3times with PBS and a substrate, ABTS, was applied. The optical densityof the samples were read at 405 nm.

The competitive ELISA format worked to indirectly detect free antigen insamples. There was a correlation between increasing amounts of antigenin the samples and reduced optical density values. Further experimentswere conducted to demonstrate that this relationship remained intactwhen varying the amount of the labeled or unlabeled antigen in the testsample and to demonstrate the importance of coating and blocking wellsprior to sample incubations. See FIGS. 8 and 9.

EXAMPLE 8 Assay Optimization

The antibody detection ELISA may be optimized for diagnosis of visceraland cutaneous leishmaniasis. For example, for assay optimization for VL,the antigens released by L. donovani promastigotes (WHO Reference Strain065) in vitro at 26° C. for 72 hours into serum-free and protein-freemedium were obtained as described in Martin et al., (1998) and used. Thesoluble antigens were separated by SDS-PAGE using 4-15% Novex gradientgels (Novex, San Diego, Calif.). The proteins were visualized bycolloidal Coomassie blue G-250 and silverstaining. The molecular weightsof the secreted antigens were estimated with the reference to aprestained standard (Novex, San Diego, Calif.).

Titration and checker-board analyses were performed to optimize theassay protocol. Optimal results were obtained when antigen (50 μg/ml)was coated with PBS-methyl glyoxal buffer and the wells were blockedwith 0.5% casein. It was found that a serum dilution of 1:500 inantigen-coated wells blocked with 0.5% casein generated lowestabsorbance with negative control sera and higher absorbance withpositive sera, sera from well-characterized, culture positive casesubjects (used as reference positives).

An equal number of sera from North American naives with no travelhistory to leishmania endemic areas were used as reference negatives,negative controls or negative sera. These sera samples were used asreference samples to optimize the assay. The reference sera were notpooled and were used as individual data points. After optimizing theassay, individual sera samples obtained from endemic areas of N. Africawere screened and used to show assay performance in terms of specificityand sensitivity.

Two ELISA plates, Greiner (Greiner Labortecnik ELISA plate Cat # 705071)and Labsystem (Labsystem combiplate breakable 8 Cat # 95029400), wereevaluated.

The following four different coating buffers (a) 0.2Mcarbonate/bicarbonate buffer pH 9.6, (b) 0.01M phosphate buffer (PO₄) pH7.2, (c) blank culture medium (serum-free) pH 7.2, and (d) 0.1Mphosphate buffered saline (pH 7.2)+1% methyl glyoxal were tested fortheir ability to immobilize the antigen onto the solid phase ELISAwells.

Gelatin ranging in concentration from 0.1 to 0.4% solution and 0.5%casein were tested. BSA and other routinely employed blocking agentswere not tested because of non-specific binding noted in previousexperiments.

PBS/T and PBS/T+0.2M NaCl were used as the washing buffers. An automaticplate washer (Denley Well Wash 04) with 4 cycle wash in each step wasused to wash the ELISA wells. In most of the assays the wash buffer wasused as the diluent for sera as well as the conjugated detectorantibody.

Two anti-human IgG conjugated to HRP (HRP conjugate) were tested. A goatanti-human IgG-HRP (Kirkegaard & Perry Laboratories, Gaithersburg, Md.),a polyclonal conjugate (PAb conjugate), and a mouse anti-human IgG-HRP(Cellabs, Brookvale, Australia), (MAb conjugate).

All steps of the ELISA were performed at room temperature. The S/Nratio, the differential absorbance between the negative sample versusthe test or positive sera sample, was used to quantify the specificantigen and antibody reactions. An anti-human monoclonal antibodyconjugated with HRP (MAb conjugate) outperformed a commerciallyavailable anti-human polyclonal antibody conjugate (PAb conjugate) (Cellabs Pty, Ltd. Sidney, Australia). The MAb conjugate gave minimalbackground reactions with endemic sera.

Generally, the wells of-an ELISA plate were coated with 50 μg/mL of thesoluble antigens released by L. donovani promastigotes in serum-free andprotein-free medium mixed in PBS-methyl-glyoxal overnight. Afterremoving the antigen, the wells were blocked with 0.5% casein for 1 hourat room temperature. Test sera along with positive and negative controlsera diluted 1:500 in PBS/T, were reacted for 1 hour at roomtemperature. After washing the plate with PBS/T, the wells were reactedwith an HRP-conjugated anti-human antibody, the detector antibody, for40 minutes at room temperature. The plates were washed and specificbinding of antigens on the solid phase and the specific antibodiespresent in the test sera were measured indirectly by the binding of HRPlabeled detector antibody which was further detected by using TMB+H₂O₂as a chromogenic substrate.

TMB solution A and B (KPL, Gaithersburg, Md.) were mixed in equal parts5-10 minutes before and transferred an aliquot of 100 μl to each well asper the guidelines provided by the manufacturer. During thedevelopmental phase of the assay, the color intensity of positive serawells was monitored (absorbance OD of 620 nm) and stop solution wasadded when those wells reached an OD of 0.450. In the final optimizedassay, the incubation time with substrate was fixed at 25 minutes.

After adding the stop solution (1M Phosphoric acid prepared in distilledwater) the contents were mixed and the plate was read at dual filter(450/620 nm) using a plate reader (Anthos LabTec Instruments 2001).

The raw data from the plate, i.e., absorbance at 450/620 nm, wereplotted into histograms and graphs. The relative specific binding wasquantified by the signal to noise (S/N) ratio which was calculated bydividing the mean absorbance, i.e., absorbance at 450/620 nm, of testsera with the mean absorbance of negative sera, and plotted. The S/Nratio was directly proportional to the specific antibody reactivity inELISA. A batch of n=22 endemic sera from North Africa were evaluated andresulted with 100% specificity and sensitivity, 99.99% PPV (positivepredictive values) and 95.45% NPV (negative predictive values).

The LabSystem plate did not perform well in this study. There was nodiscrimination between the positive and negative sera. There was a cleardistinction between sera in the Greiner plate. It was found that thePBS+methyl glyoxal, phosphate buffer and culture medium respectivelyshowed higher reactivity with positive sera and relatively lessreactivity with negative sera. A higher S/N ratio was seen using sera ata 1:500 sera dilution. Phosphate buffer, culture medium and PBS-glyoxalgave higher S/N ratios. Therefore, the Greiner plate and PBS+methylglyoxal were selected as ELISA plate and coating buffer respectively.

Using the Greiner ELISA plate and PBS+methyl glyoxal as the coatingbuffer for the test, positive and negative sera were used at 1:500dilution. Two anti-human IgG-HRP conjugates were evaluated at 4different dilutions. The PAb conjugate was found to be highly reactivewith the negative sera indicating a high level of non-specific reaction.On the other hand, the same level of reactivity was observed in theblank and negative sera sample wells with the MAb conjugate. Thereactivity with MAb conjugate appeared to be more specific with positivesera as evidenced by higher S/N ratios.

Negative and positive sera were reacted at 1:500 dilution and washedwith two different wash solutions. MAb conjugate was used at 1:8000dilution. Plates were read at 450/620 nm. Reactivity of blanks andnegative sera were lower in wells washed with PBS/T+0.2M NaCl. The dataindicated that PBSIT+0.2M NaCl was more effective in removingnon-specific binding (nearly 45% reduction of non-specific signal 0.283v. 0.114) and increasing the S/N ratio from 3.7 to 7.2. Despite itshigher S/N ratio, 0.2M NaCl was left out of the final wash bufferbecause it formed a precipitate on standing.

Having selected the Greiner plate and PBS+glyoxal as the coating buffer,optimum levels of antigen and appropriate blocking reagents wereinvestigated. Wells were coated with a series of antigen concentrationfrom 1.25 ug/ml to 40 ug/ml. Two blocking reagents, 0.4% gelatin and0.5% casein prepared in distilled water were evaluated. Positive andnegative sera, diluted 1:500 in PBS/T, reacted in the Ag-coated andblocked wells for 1 hour at room temperature. Two conjugates, PAb andMAb-conjugates, diluted 1:4000 in PBS/T were added to wells andincubated for 30 minutes at room temperature. The color was developedfor 25 minutes by adding the substrate and immediately read afteraddition of stopping solution. The reactivity was higher in blank andnegative sera wells with PAb conjugate thereby reducing the differencesbetween samples that resulted with a low SIN ratio. With the MAbconjugate, the absorbance of blank and negative sera wells was almostequal. There was a pattern in the reactivity relative to the antigenconcentration. Wells reacted with the positive sera showed a gradualrise in absorbance dependent upon the antigen concentration. On thewhole, MAb conjugate reactivity was relatively lower in control samplewells. The S/N ratios were higher with MAb conjugate when casein usedfor blocking. MAb conjugate with casein blocking generated excellent S/Nratios, at 20 and 40 μg/ml antigen levels.

Experiments involving the relative kinetics of antibody reactivity atdifferent sera dilutions provided a good discrimination at 1:500dilution. This formed the basis for future assays.

After optimizing assay steps, the following protocol was followed forevaluating test sera samples of subjects from endemic areas. In short,the wells were coated with 50 μg/ml soluble antigen mixed inPBS-methyl-glyoxal buffer overnight and after removing the antigen, thewells were blocked with 0.5% casein for 1 hour at room temperature. Testserum along with control sera diluted 1:500 in PBS/T, was reacted for 1hour at room temperature. After washing the plate in PBS/T, the wellswere reacted with MAb conjugate at 1:8000 dilution for 40 minutes atroom temperature and after washing, the TMB substrate was added andcolor development was allowed to proceed for 25 min and then stoppedwith the stop solution.

Absorbance was read at dual filter (450/620 nm) and the results wereanalyzed. A total of n=22 test clinical sera obtained from the endemicareas of North Africa were evaluated along with n=5 reference controlnegative sera.

SDS-PAGE analysis was conducted Coomassie staining showed several majorbands with approximate molecular weights of 11, 30, 42, 50 and 161 kDa.In addition to these abundant bands, silver staining revealed moredistinct protein bands of approximately 6, 15, 17, 22, 58, and 107 kDa.This illustrates that the test contained a variety of protein antigens.

The cut-off value in the current assay was mean+3 SD of negative (n=5)sera (Mean 0.1304, SD=0.042), i.e., 0.278 which is rounded off to 0.300.Using an absorbance OD450/620 nm of 0.300 as the cut off, n=22 test serafrom field were categorized as either positive or negative. With theexception of one sample, all were positive. The sensitivity,specificity, PPV and NPV were calculated. Thus, the sensitivity andspecificity were both 100%, the PPV was 99.9% and NPV was 95.45%. Theresultant S/N ratio of these samples suggests that the assay is highlysensitive and specific.

Clearly, one of ordinary skill in the art may further optimize theassays of the invention by changing various assay conditions by methodsstandard in the art.

Incorporation by Reference

To the extent necessary to understand or complete the disclosure of thepresent invention, all publications, patents, and patent applicationsmentioned herein are expressly incorporated by reference therein to thesame extent as though each were individually so incorporated.

1-33. (canceled)
 34. A protein-free medium comprising an agent thatbalances the oncotic pressure across a semi-permeable cell membrane. 35.The protein-free medium of claim 34, wherein the agent is D, xylose. 36.The protein-free medium of claim 34, further comprising at least one ofthe following ingredients: Hepes buffer, L-glutamine and sodiumbicarbonate without phenol red. 37-72. (canceled)
 73. The protein-freemedium of claim 34, comprising RPMI Medium 1640, 0.076 mM D, xylose, 25mM Hepes buffer, L-glutamine, and 30 mM sodium bicarbonate withoutphenol red.
 74. A protein-free medium comprising RPMI Medium 1640, 0.076mM D, xylose, 25 mM Hepes buffer, L-glutamine, and 30 mM sodiumbicarbonate without phenol red.
 75. A protein-free medium XOM™ availablefrom Invitrogen/GIBCO BRL as formula number 96-0051 DJ.